Intergenic suppression of the gammaM23K uncoupling mutation in F0F1 ATP synthase by betaGlu-381 substitutions: the role of the beta380DELSEED386 segment in energy coupling.
We previously demonstrated that the Escherichia coli F0F1-ATP synthase mutation, gammaM23K, caused increased energy of interaction between gamma- and beta-subunits which was correlated to inefficient coupling between catalysis and transport [Al-Shawi, Ketchum and Nakamoto (1997) J. Biol. Chem. 272, 2300-2306]. Based on these results and the X-ray crystallographic structure of bovine F1-ATPase [Abrahams, Leslie, Lutter and Walker (1994) Nature (London) 370, 621-628] gammaM23K is believed to form an ionized hydrogen bond with betaGlu-381 in the conserved beta380DELSEED386 segment. In this report, we further test the role of gamma-beta-subunit interactions by introducing a series of substitutions for betaGlu-381 and gammaArg-242, the residue which forms a hydrogen bond with betaGlu-381 in the wild-type enzyme. betaE381A, D, and Q were able to restore efficient coupling when co-expressed with gammaM23K. All three mutations reversed the increased transition state thermodynamic parameters for steady state ATP hydrolysis caused by gammaM23K. betaE381K by itself caused inefficient coupling, but opposite from the effect of gammaM23K, the transition state thermodynamic parameters were lower than wild-type. These results suggest that the betaE381K mutation perturbs the gamma-beta-subunit interaction and the local conformation of the beta380DELSEED386 segment in a specific way that disrupts the communication of coupling information between transport and catalysis. betaE381A, L, K, and R, and gammaR242L and E mutations perturbed enzyme assembly and stability to varying degrees. These results provide functional evidence that the beta380DELSEED386 segment and its interactions with the gamma-subunit are involved in the mechanism of coupling.
Ketchum
C J
CJ
Department of Molecular Physiology and Biological Physics, University of Virginia, P.O. Box 10011, Charlottesville, VA 22906-0011, USA.
Al-Shawi
M K
MK
Nakamoto
R K
RK
D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D018360:Crystallography, X-Ray; D004734:Energy Metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013489:Suppression, Genetic; D013816:Thermodynamics
D018698:Glutamic Acid; D001120:Arginine; D006180:Proton-Translocating ATPases